Mapping Intercellular Protein Trafficking in Glioblastoma with Esterase-Activated Proximity Labeling

Project Summary Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults with an average survival of 15-18 months. The heterogeneous nature of GBM is complicated by the presence of diverse cancer and non-cancer cells within the tumor. The intercellular crosstalk, including protein translocation, in GBM promotes chemoresistance, invasion, and angiogenesis and leads to a dismal prognosis. Despite extensive cancer biology research, a detailed molecular picture of protein trafficking between GBM and other non-cancer cell types remains largely unclear. This proposal aims to develop a new molecular method for mapping translocated proteins between GBM cells and other brain cell populations in an in vivo setting. This work is built upon an in-vivo compatible proximity labeling enzyme LipoID, which I developed early in my postdoctoral career. LipoID could employ unnatural substrates containing a click reaction handle to label the living glioblastoma proteome. In order to distinguish resident and translocated proteins within the destination non-cancer cells, this proposal aims to develop a new esterase-activated bioorthogonal reaction that allows for stepwise proteome labeling in two different cell types (Aim1). This method will enable the precise recording of protein translocation with cell-specificities in both GBM cells and non-cancer cells. In Aim2, systematic profiling of intercellular protein trafficking within the GBM microenvironment will be performed in both in vitro co-culture system (Aim2.1), and in vivo syngeneic models (Aim2.1). Quantitative protein mass spectrometry will be used identify significant protein translocation events between GBM and other brain cell types (neurons, astrocytes, microglia, and endothelial cells). To functionally validate the importance of the top proteomic targets, Aim2.3 will focus on creating (conditionally) knock out GBM cells for syngeneic GBM models generation. Tumor growth and mice survival will be measured and compared with the effect of Wildtype GBM cells. This study could open exciting avenues of research in understanding gliomagenesis, and potentially unveil new targets for GBM therapeutic development. The esterase-activated click reaction may find broader usage in biomedical research by providing spatial and temporal resolution for bioconjugation. My mentor and collaborators possess extensive expertise in molecular tool development, brain cancer biology, quantitative proteomics, bioorthogonal chemistry, and glioblastoma mouse models, thereby equipping me with the necessary training to execute the proposed research. Additionally, they will offer mentorship to acquire all requisite professional skills and preliminary data for a successful transition to my independent cancer research lab. There, I aim to dedicate my career to unraveling cancer-causing mechanisms and developing innovative cancer therapies.